Very little is known about the way proteins fold in the cellular environment. More specifically, the degree of folding and molecular shape achieved by ribosome-bound nascent proteins is largely unexplored by methodologies able to provide a direct assessment of protein conformation. The goal of this research is to investigate the short-range helical secondary structure and degree of hydrophobic collapse (or lack thereof) of ribosome-bound nascent proteins in the absence and presence of the trigger factor (TF) chaperone. Different stages of nascent chain elongation will be examined. Nascent chains derived from the three proteins apomyoglobin, apoHmpH and Fim H will be analyzed. Local secondary structure and, most importantly, hydrophobic collapse are two well known major driving forces for protein folding in vitro. However, nothing is known about their importance in the context of folding as proteins emerge out of the ribosome. This project will be primarily carried out by Forster resonance energy transfer (FRET) via fluorescence lifetime measurement of the FRET donor in the absence and presence of the acceptor. We will assess FRET efficiency variations (proportional to variations in intra-molecular distance distributions) to monitor changes in secondary and tertiary structure of ribosome-bound model proteins as the nascent chains emerge out of the ribosomal tunnel to gain evidence about their degree of local structure and collapse. The specific dependence of the above properties on the presence and absence of the TF chaperone will also be investigated, in light of the fact that the TF nonpolar inner surface may effectively bind the nascent incomplete proteins and dramatically alter their structure and degree of compaction.